The effect on antibody heavy chain dimerization of complementary charged amino acids engineered on the CH3 domains was investigated. The T366 and Y407 located on the CH3 domain interface of two antitumor antibodies, CC49IL2 (CC49 with an interleukin-2 attached to its carboxyl end) and COL-1, were replaced with arginine and glutamic acid residues, respectively. The possible attraction of the complementary charges to form a heterodimer was detected by simultaneously expressing the engineered CC49IL2 and COL-1 constructs in Spodoptera frugiperda (Sf9) insect cells. Among the combinations studied, the Y407R / T366E combination yielded the highest amount of heterodimer which is about 68% of the total dimers produced compared to about 30% and 33% for the other two combinations, T366R / Y407E and T366R:Y407R / T366E:Y407E, respectively. Modeling of the engineered mutations showed that two H-bonds could form between R407 on CC49IL2 chain and E366 on COL-1 which could explain why the combination Y407R / T366E resulted in more of the heterodimer than the other two combinations studied. These results suggest that pairing of complementary charges engineered on CH3 region can result in the attraction of different antibody chains favoring formation of a heterodimer or a bispecific antibody.